The present invention relates to new amidinophenylpyruvic acid derivatives antagonistic to activated blood coagulation factor VII (FVIIa) and pharmaceutically acceptable salts thereof. The amidinophenylpyruvic acid derivatives and salts thereof of the present invention are usable as active ingredients of blood anticoagulants, pharmaceutical composition for preventing or treating a thrombus or embolus, intimal thickening inhibitors or pharmaceutical composition for preventing or treating angiostenosis and vascular occlusion.
When a thrombus or embolus is formed in a blood vessel, the normal blood flow becomes impossible to cause various diseases. The anticoagulation therapy is one of internal medical treatment methods for treating and preventing thrombosis like fibrinolytic therapy and antiplatelet therapy.
Anticoagulants available on the market are warfarin capable of inhibiting the formation of coagulation factors, thrombin inhibitors, etc. However, they often have undesirable side effects such as causing of serious hemorrhage and, therefore, they cannot easily control of the coagulating property. Now, it is demanded in the art to find a compound having an anticoagulating activity based on a new function mechanism and free of the undesirable side effects of the existing anticoagulants. It is also demanded in the art to find an anticoagulating compound having a high peroral activity and free of undesirable side effects.
The blood coagulation paths are roughly divided into intrinsic path and extrinsic path. Although the relative importance of each of the two coagulation paths has not yet been elucidated, it is said that the extrinsic coagulating reaction is deeply concerned in the acceleration of coagulation. The origin of the extrinsic coagulating reaction is the incidence of glycoprotein tissue factor (TF) on the surface of a cell membrane. When FVIIa and TF form a complex (FVIIa/TF) together, the activation is further accelerated. The complex having serine protease-type activity specifically reacts on blood coagulation factors X and IX to activate the respective factors. As a result, thrombin is activated to form a thrombus. The term xe2x80x9cinhibition of activated blood coagulation factor VII (FVIIa inhibition)xe2x80x9d herein indicates the inhibition of the activity of FVIIa/TF complex enzyme.
Thus, compounds having the FVIIa inhibition activity have an excellent blood anticoagulating activity, and they will be possibly used as medicines free of the defects of ordinary anticoagulants. Further, medicines capable of selectively inhibiting FVIIa will be possibly used as excellent anticoagulating medicines substantially free from side effects.
Disseminated intravascular coagulation is usually caused because a tissue factor forms a complex with FVIIa in the blood, and an FVIIa inhibitor is possibly particularly effective against generalized intravascular blood coagulation syndrome.
It is known that thrombi are hardly formed in the blood of patients lacking in VII factor at a high shear rate. The FVIIa inhibitor is possibly effective in controlling the thrombus formation particularly in narrowed arteries (R. Bastad et al., Blood. 84, 3371 (1994)).
The FVIIa inhibitor is possibly effective in controlling the restenosis after angioplasty with, for example, a balloon catheter (Y. Jang et al., Circulation 92, 3041 (1995) and D. W. Courtman et al., Circ Res. 82, 996 (1998)).
It is known that FVIIa/TF causes the migration of smooth muscle cells of blood vessels. The activity of a compound to inhibit this effect can be examined by a method described in Sato Y et al., Thromb Haemost. 78, 1138 (1997). FVIIa inhibitor might be effective in not only inhibiting the thrombus formation but also controlling the thickening of intima by inhibiting the migration of smooth muscles to control the narrowing or occlusion of the blood vessels.
However, compounds clinically usable as FVIIa inhibitor have not been obtained yet.
An object of the present invention is to provide compounds having an excellent effect of inhibiting the action of activated blood coagulation factor VII.
Another object of the present invention is to provide compounds capable of selectively inhibiting activated blood coagulation factor VII.
A third object of the present invention is to provide peroral compounds selectively inhibiting the action of activated blood coagulation factor VII.
A fourth object of the present invention is to provide a pharmaceutical composition.
A fifth object of the present invention is to provide a pharmaceutical composition for preventing or treating diseases caused by the blood coagulation, thrombus or embolus, intimal thickening or angiostenosis.
A sixth object of the present invention is to provide a pharmaceutical composition for preventing or treating disseminated intravascular coagulation, deep vein thrombosis, diseases caused by pulmonary vascular disorder, diseases caused by an ischemic heart disease, diseases caused by a cerebrovascular disorder, occlusion of blood vessel and angiostenosis after an operation for forming a bypass in coronary artery, coronary artery intervention after percutaneous transluminal coronary angioplasty (PTCA), occlusion of blood vessel and angiostenosis after percutaneous transluminal coronary recanalization (PTCR), formation of thrombi after artificial blood vessel-forming operation or artificial valve replacement, peripheral embolism, formation of thrombi in the course of the extracorporeal circulation and antiphospholipid antibody syndrome.
A seventh object of the present invention is to provide a pharmaceutical composition for preventing or treating cerebral infarction or cerebral stroke.
A eighth object of the present invention is to provide an antagonist against activated blood coagulation factor VII (FVIIa).
Under these circumstances, the inventors made investigations on FVIIa inhibiting activity of benzamidine derivatives disclosed in WO 98/31661 as compounds having an effect of inhibiting activated blood coagulation factor X (FXa). As a result, the inventors found that only specified amidinophenylpyruvic acid derivatives have a high activity of inhibiting FVIIa. After synthesizing the new amidinophenylpyruvic acid derivatives and examining the effect thereof on extrinsic blood coagulation factors on the basis of these facts, the inventors have found that they are highly useful new compounds having an activation profile different from that of ordinary ones. Namely, these compounds have a high activity of inhibiting FVIIa and a high selectivity toward thrombin. The present invention has been completed on the basis of these findings.
The present invention provides amidinophenylpyruvic acid derivatives of the following general formula (1) or (1-2), pharmaceutically acceptable salts thereof and an antagonist against activated blood coagulation factor VII (FVIIa), which contains such a compound(s) as the active ingredient: 
wherein W represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms,
X represents a hydrogen atom, a carboxyalkyl group having 2 to 5 carbon atoms, or
a methyl or ethyl group having a substituent(s) selected from the group consisting of alkoxycarbonyl groups having 2 to 8 carbon atoms, carbamoyl group, tetrazolyl group, sulfo group, sulfamoyl group, phosphono group and hydroxyl group,
a benzyl group which may have a substituent(s) selected from the group consisting of hydroxyl group, carboxyl group, tetrazolyl group, sulfo group, sulfamoyl group, phosphono group, halogeno groups and alkyl groups having 1 to 3 carbon atoms, or
an alkyl group having 1 to 4 carbon atoms, phenyl group, guanidinopropyl group, mercaptomethyl group, imidazolylmethyl group, aminobutyl group, aminopropyl group, (methylthio)ethyl group or indolylmethyl group, X and W may be bonded together to form a ring, and in this case, xe2x80x94Wxe2x80x94Xxe2x80x94 represents ethylene group, trimethylene group or tetramethylene group, V represents any of the following groups (1) to (8):
(1) benzimidazolecarbonyl, quinolinecarbonyl, benzothiazolecarbonyl or benzoxazolecarbonyl group which may have a substituent(s) selected from the group consisting of amino group, alkyl groups having 1 to 6 carbon atoms, halogeno groups and alkoxyl groups having 1 to 6 carbon atoms,
(2) benzoyl group substituted with phenyl, pyridyl or thiophenyl group having a substituent(s) selected from the group consisting of amidino group, guanidino group, amino group, dialkylamino groups having 2 to 5 carbon atoms, carboxyl group and acyl groups having 1 to 6 carbon atoms,
(3) benzoyl group substituted with phenoxymethyl or benzoylamino group which may have a substituent(s) selected from the group consisting of amidino group, methoxyl group, guanidino group, amino group and dialkylamino groups having 2 to 5 carbon atoms,
(4) guanidinocarbonylbenzoyl group or (guanidinomethyl)benzoyl group,
(5) a monoalkylguanidinobenzoyl group having 9 to 11 carbon atoms wherein the alkyl group bonded to the guanidino group may be also bonded to benzoyl group to form a ring,
(6) naphthalenecarbonyl, thiophenecarbonyl or 1-naphthalenesulfonyl group which may have a substituent(s) selected from the group consisting of guanidino group, guanidinocarbonyl group, guanidinomethyl group and amidino group,
(7) benzoyl group which may have a substituent(s) selected from the group consisting of hydroxyl group, N-methylpyrrolidyloxy group, pyrrolidylmethyl group, imidazolylmethyl group and aminoimidazolylmethyl group, and
(8) dihydrobenzofurancarbonyl or dihydrobenzopyrancarbonyl group which may have a substituent(s) selected from the group consisting of amidino group, guanidino group, amino group, alkyl groups having 1 to 6 carbon atoms, halogeno groups and aryl groups having 4 to 6 carbon atoms,
groups (1) to (7) described above may further have an alkoxyl group having 1 to 6 carbon atoms on an aromatic carbon at the o-position to the carbonyl group or sulfonyl group bonded to N in general formula (1), and when the alkyl group on the alkoxyl group has 2 or 3 carbon atoms, it may be bonded to a carbon atom on the aromatic ring to form a ring,
Y represents oxygen atom or sulfur atom, and
Z represents hydroxyl group or amino group which may have a substituent(s) selected from the group consisting of phenyl group, benzyl group and phenethyl group, or 
wherein X2 represents hydrogen atom, carboxymethyl group or carboxyethyl group, and
V2 represents benzoyl group having a substituent(s) selected from the group consisting of alkoxyl groups having 1 to 5 carbon atoms at the o-position to the carbonyl group in benzoyl group,
iodine atom or phenyl, thiophenyl, pyridyl or guanidino group at the m-position to the carbonyl group in benzoyl group, or
guanidino or amino group at the p-position to the carbonyl group in benzoyl group, or
V2 represents 1-(4-pyridyl)piperidine-3-carbonyl group or indole-5-carbonyl group.
In general formula (1), W is preferably hydrogen atom or an alkyl group having 1 to 3 carbon atoms. Hydrogen is particularly preferred.
In general formula (1), Y is preferably oxygen atom.
In general formula (1), preferably, W is hydrogen atom and Y is oxygen atom.
In general formula (1), the carboxyalkyl group X having 2 to 5 carbon atoms is preferably that having 2 to 4 carbon atoms. The carboxyalkyl group X is more preferably carboxymethyl group or carboxyethyl group. X is preferably hydrogen atom, carboxymethyl group or carboxyethyl group. X is more preferably carboxyethyl group, and in particular, (R)-carboxyethyl group.
In general formula (1), V is preferably benzoyl group having a substituent(s) derived from guanidino group, benzoyl group having an aryl group having a substituent(s) or phenoxymethyl group having a substituent(s) at the 3-position or a group composed of benzoyl group condensed with a five-membered or six-membered aromatic heterocyclic ring.
An example wherein an alkoxyl group having 1 to 6 carbon atoms in the groups of above formulae (1) to (7) is bonded to an aromatic carbon at the o-position to carbonyl group bonded to N in general formula (1) is 2-Methoxy-3-(4-amidinophenyl)benzoyl group when V is 3-(4-amidinophenyl)benzoyl group.
V in general formula (1) is preferably as follows:
Benzimidazolecarbonyl, quinolinecarbonyl or benzothiazolecarbonyl group which may have a substituent(s); the substituent being selected from the group consisting of amino group, alkyl groups having 1 to 6 carbon atoms, halogeno groups and alkoxyl groups having 1 to 6 carbon atoms;
Benzoyl group substituted with phenyl, pyridyl or thiophenyl group, having a substituent(s), at the 3-position thereof; the substituent being amidino group, guanidino group or amino group;
Benzoyl group substituted with phenoxymethyl group, having a substituent(s), at the 3-position thereof; the substituent being amidino group, guanidino group or amino group;
Guanidinocarbonylbenzoyl group or (guanidinomethyl)benzoyl group;
Naphthalenecarbonyl or thiophenecarbonyl group having a substituent(s): the substituent being guanidino group, guanidinocarbonyl group or guanidinomethyl group; or
A monoalkylguanidinobenzoyl group having 9 to 11 carbon atoms, wherein the alkyl group bonded to guanidino group may be also bonded to benzoyl group to form a ring.
V in general formula (1) is preferably as follows:
Benzimidazole-5-carbonyl, quinoline-6-carbonyl or benzothiazole-6-carbonyl group which may have a substituent(s); the substituent being an amino group(s), an alkyl group(s) having 1 to 6 carbon atoms, a halogeno group(s) or an alkoxyl group(s) having 1 to 6 carbon atoms;
Benzoyl group substituted with phenyl, pyridyl or thiophenyl group, having a substituent(s), at the 3-position thereof; the substituent being amidino group or guanidino group;
Benzoyl group substituted with phenoxymethyl group, which may have a substituent(s), at the 3-position thereof; the substituent being amidino group or guanidino group;
3-Guanidinocarbonylbenzoyl group or 3-guanidinomethylbenzoyl group; or
1-Amidino-1,2,3,4-tetrahydroquinoline-6-carbonyl group.
V in general formula (1) is more preferably as follows:
Benzimidazole-5-carbonyl, quinoline-6-carbonyl or benzothiazole-6-carbonyl group which may have a substituent(s); the substituent being an amino group(s) or an alkyl group(s) having 1 to 3 carbon atoms,
Benzoyl group substituted with phenyl or pyridyl group, having a substituent which is amidino group or guanidino group, at the 3-position;
3-Guanidinocarbonylbenzoyl group or
1-Amidino-1,2,3,4-tetrahydroisoquinoline-6-carbonyl group.
In general formula (1), V is particularly preferably benzothiazole-6-carbonyl group.
In general formula (1), Z is preferably hydroxyl group.
Preferred amidinophenylpyruvic acid derivatives of the above formula (1) and pharmaceutically acceptable salts thereof are those of general formula (1) wherein:
W represents hydrogen atom or an alkyl group having 1 to 3 carbon atoms,
X represents hydrogen atom or a carboxyalkyl group having 2 to 5 carbon atoms,
V represents benzimidazolecarbonyl, quinolinecarbonyl or benzothiazolecarbonyl group which may have a substituent(s); the substituent being an amino group(s), an alkyl group(s) having 1 to 6 carbon atoms, a halogeno group(s) or an alkoxyl group(s) having 1 to 6 carbon atoms,
benzoyl group substituted with phenyl, pyridyl or thiophenyl group having a substituent, at the 3-position thereof, the substituent being amidino group, guanidino group or amino group,
benzoyl group substituted with phenoxymethyl group having a substituent(s), at the 3-position thereof, the substituent being amidino group, guanidino group or amino group,
guanidinocarbonylbenzoyl group or (guanidinomethyl)benzoyl group, naphthalenecarbonyl or thiophenecarbonyl group having a substituent(s);
the substituent being guanidino group, guanidinocarbonyl group or guanidinomethyl group, or
a monoalkylguanidinobenzoyl group having 9 to 11 carbon atoms, wherein the alkyl group bonded to guanidino group may be also bonded to benzoyl group to form a ring, and
Z represents hydroxyl group.
More preferred compounds are as follows:
compounds of general formula (1) wherein X is limited to a group listed above to be preferred as X,
compounds of general formula (1) wherein Y and W are each limited to a group listed above to be preferred, and
compounds of general formula (1) wherein V is limited to a group listed above to be preferred.
Further, the following compounds of general formula (1) are preferred:
W represents hydrogen atom,
X represents hydrogen atom or carboxyethyl group,
V represents benzimidazole-5-carbonyl, quinoline-6-carbonyl or benzothiazole-6-carbonyl group which may have a substituent(s); the substituent being amino group or an alkyl group having 1 to 3 carbon atoms,
Y represents oxygen atom, and
Z represents hydroxyl group.
Preferred amidinophenylpyruvic acid derivatives of the above formula (1) and pharmaceutically acceptable salts thereof are those wherein:
W represents hydrogen atom,
X represents hydrogen atom or carboxyethyl group,
V represents 3-guanidinocarbonylbenzoyl group, 3-guanidinomethylbenzoyl group, 3-(4-amidinophenoxymethyl)benzoyl group, benzimidazole-5-carbonyl group, 2-methylbenzimidazole-5-carbonyl group, 2-aminobenzimidazolecarbonyl group, 2-aminoquinoine-6-carbonyl group, 3-(4-amidinophenyl)benzoyl group, 3-(4-guanidinophenyl)benzoyl group, 1-amidino-1,2,3,4-tetrahydroquinoline-6-carbonyl group or 2-aminobenzothiazole-6-carbonyl group,
Y represents oxygen atom, and
Z represents hydroxyl group.
Preferred amidinophenylpyruvic acid derivatives of the above formula (1) and pharmaceutically acceptable salts thereof are those wherein:
W represents hydrogen atom,
X represents carboxyethyl group,
V represents 2-methylbenzimidazole-5-carbonyl group, 2-amiobenzimidazole-5-carbonyl group, 2-aminoquinoline-6-carbonyl group or 2-aminobenzothiazole-6-carbonyl group,
Y represents oxygen atom and
Z represents hydroxyl group.
In general formula (1-2),
X2 is preferably carboxyethyl group or hydrogen atom, and when V2 represents benzoyl group having a substituent(s), the substituent is preferably 3-phenyl group, 2-isopropoxy group, 2-cyclopentyloxy group or 4-guanidino group.
Compounds of general formula (1) or (1-2) are preferably those described in Examples, though the compounds are not limited to them. Compounds described in Examples 1, 2, 3, 4, 13, 27, 28 and 39 are preferred. The compound described in Example 28 is particularly preferred.
The alkyl groups and alkoxyl groups in the compounds of the present invention may be branched or they may have a ring. For example, alkyl groups include, for example, isopropyl group, and alkoxyl groups include, for example, cyclobutyloxy group. In dialkylamino groups, two alkyl groups may be bonded to each other to form a ring such as 1-pyrrolidyl group. The halogeno groups include fluorine atom, chlorine atom and bromine atom. In the monoalkylguanidinobenzoyl groups, the alkyl group may be bonded to guanidino group and benzoyl group to form a ring. In this case, an aromatic hydrocarbon at the o-position to guanidino group is bonded to one of the nitrogen atoms in guanidino group through methylene, ethylene or triethylene group. Examples of these groups are 1-amidino-1,2,3,4-tetrahydroquinolinecarbonyl group and 2-amino-3,4-dihydroquinazolinecarbonyl group.
The compounds of the present invention include mixtures of various stereoisomers such as geometrical isomers, tautomers and optical isomers, as well as isolated ones. Amidino group in the compounds of the present invention may be replaced with a suitable substituent capable of being changed into the amidino group in vivo. For example, in general formulae (1) and (1-2), hydrogen atom bonded to nitrogen atom having double bond in amidino group bonded to benzene ring may be replaced with hydroxyl group, an alkoxyl group such as ethoxyl group, amino group, carboxyl group, an alkoxycarbonyl group such as ethoxycarbonyl group, an alkylsulfonyl group such as ethylsulfonyl group, carbamoyl group, a carbamoyl group in which one or two hydrogen atoms are replaced with an alkyl group such as diethoxycarbamoyl group, formyl group, an acyl group such as acetyl group and an alkylcarboxyl group such as acetoxyl group.
The salts of amidinophenylpyruvic acid derivatives represented by general formula (1) or (1-2) are pharmaceutically acceptable salts such as salts thereof with mineral acids, e.g. hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid and phosphoric acid; salts thereof with organic acids, e.g. formic acid, acetic acid, lactic acid, salicylic acid, mandelic acid, citric acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, tannic acid, malic acid, tosyl acid, methanesulfonic acid and benzenesulfonic acid; salts thereof with alkali metals, alkaline earth metals and alkylammonium groups, e.g. sodium, potassium, calcium, ammonium and triethylammonium group; and hydrates thereof.
Typical processes for producing the compounds of the present invention will be described below.
An amino acid (2) in which nitrogen atom is protected with t-butoxycarbonyl group or the like can be synthesized from a well-known amino acid derivative by an ordinary method. The amino acid (2) is subjected to a proper reduction reaction such as a reaction with ethyl chloroformate in the presence of, for example, a base to activate carboxyl group and to form a mixed acid anhydride, followed by the treatment with NaBH4. Thus an alcohol compound (3) can be obtained. The alcohol compound (3) is reacted with, for example, carbon tetrabromide in the presence of triphenylphosphine or with methanesulfonyl chloride in the presence of a base to obtain a compound (4) having a leaving group such as bromine atom, methanesulfonyloxy group or chlorine atom in place of hydroxyl group. 
wherein Prot represents a protecting group, Xxe2x80x2 is the same as X or it represents a group obtained by protecting a functional group in X, and L represents a leaving group.
The compound (4) thus obtained is reacted with 3-hydroxy-4-iodobenzonitrile in the presence of a base such as potassium carbonate in a solvent such as dimethylformamide to form an ether compound (5).
The ether compound (5) thus obtained can be converted into an acrylic acid ester derivative (6) by condensing it by, for example, Mizorogi-Heck reaction with methyl 2-acetamidoacrylate in the presence of palladium catalyst. 
An amine (7) can be obtained by removing the protecting group on nitrogen in the acrylic acid ester derivative (6) with, for example, an acid solvent such as hydrogen chloride-containing dioxane solvent. 
In the production of a compound of general formula (1) wherein W represents an alkyl group having 1 to 6 carbon atoms, the amine (7) obtained as described above is subjected to the reductive amination with paraformaldehyde or an alkylaldehyde and sodium cyanoborohydride or the like to obtain the compound of general formula (8) wherein W represents an alkyl group having 1 to 6 carbon atoms: 
wherein W represents an alkyl group having 1 to 6 carbon atoms.
Thus obtained amine (8) wherein W represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms is condensed with a suitable carboxylic acid in the presence of a condensing agent such as dicyclohexylcarbodiimide in a solvent such as methylene chloride or dimethylformamide, or it is reacted with a suitable sulfonyl chloride or an acid halide in the presence of a base to obtain a compound (9): 
wherein Vxe2x80x2 is the same as V or it represents group V in which the functional group is protected, and W represents hydrogen atom or an alkyl group having 1 to 6 carbon atoms.
Compound (9) can be reacted with an alcohol such as ethanol or methanol in a solvent such as a solution of hydrogen chloride in dioxane to form an imidate, which is then reacted with ammonia gas or an ammonium salt such as ammonium carbonate to form a compound (10) having amidino group derived from cyano group. 
The obtained amidino compound (10) is hydrolyzed at the ester portion and enamido portion on the 2-alkoxcarbonyl-2-acetamidoethenyl group at the same time to obtain an amidinophenylpyruvic acid derivative (11) of general formula (1) wherein Z represents hydroxyl group and Y represents oxygen atom. If necessary, a step of removing the protecting group from Xxe2x80x2 or Vxe2x80x2 is inserted before or after the hydrolysis step. 
A compound of general formula (1) wherein Z represents amino group which may have a substituent(s) can be produced by, for example, Mizorogi-Hack reaction to condense an ether compound (5) with 2-acetamidoacrylic acid or the like in the presence of a palladium catalyst or by the selective hydrolysis of the ester part of the acrylic acid ester derivative (6) under an alkaline condition realized by using an aqueous sodium hydroxide solution to obtain the acrylic acid derivative (12).
Then the compound is condensed with an amine such as benzylamine in the presence of a condensing agent such as dicyclohexylcarbodiimide (DCC) to obtain a compound (13) wherein Z represents an amino group which may have a substituent(s). After the removal of the protecting group from nitrogen atom, amidation, or sulfonylation, conversion of amidino group from cyano group or hydrolysis of enamido part in the same manner as that described above, an amidinophenylpyruvic acid derivative of general formula (1) wherein Y represents oxygen atom and Z represents an amino group which may have a substituent(s) can be obtained. 
wherein Z represents amino group which may have a substituent(s).
Compounds represented by general formula (1-2) can be also produced in the same manner as that described above. The compound of general formula (1) or (1-2) thus produced or a salt thereof can be isolated by a well-known purification method such as extraction, concentration, concentration under reduced pressure, extraction with a solvent, crystallization, recrystallization, redissolution or a chromatography.
Evaluation of Antagonistic Activity to Enzymes
Determination of antagonistic activity to activated human blood coagulation factor VII:
100 nM solution of activated human blood coagulation factor VII (Enzyme Research Laboratories Ltd.) in pH 7.4 tris hydrochloride buffer solution was mixed with 100 nM solution of a tissue factor (American Diagnostica Laboratories Ltd.) in pH 7.4 tris hydrochloride buffer solution in equal amounts, and the obtained mixture was left to stand at room temperature for 30 minutes. 50 xcexcl of 50 mM tris hydrochloride buffer solution having a pH adjusted to 7.4 was added to 5 xcexcl of an aqueous solution of a compound to be tested. Then 10 xcexcl of the previously prepared mixture of 50 nM of each of the solutions of activated human blood coagulation factor VII and tissue factor was added to the obtained mixture, and they were incubated at room temperature for 10 minutes. Then 25 xcexcl of 3.2 mM solution of D-isoleucyl-L-prolyl-L-arginyl-P-nitroanilide hydrochloride (S-2288, Daiichi Pure Chemicals Co., Ltd.) in pH 7.4 tris hydrochloride buffer was added to the obtained mixture. The absorbance was determined and the initial reaction velocity was calculated. A control was prepared by using 5 xcexcl of tris hydrochloride buffer adjusted to pH 7.4 in place of the solution of the compound to be tested. The absorbance was determined with MICROPLATE READER Model 3550-UV (BIO RAD) at a wavelength of 405 nm at intervals of 5 minutes for 400 minutes. The negative logarithm (pIC50) of the concentration of the sample compound for the 50% inhibition of the activity (initial velocity) of the activated blood coagulation factor VII was determined, and it was employed as the index of the antagonistic activity to the activated blood coagulation factor VII. The antagonistic activities of typical compounds are shown in Table 1 given below.
(2) Determination of Antagonistic Activity to Human Thrombin:
130 xcexcl of 100 mM tris hydrochloride buffer adjusted at pH 8.4 was added to 10 xcexcl of an aqueous solution of a sample compound having antagonistic activity to human thrombin. Then 10 xcexcl of a solution of human thrombin (SIGMA Co. Ltd.) adjusted to 2 units/ml with pH 8.4 tris hydrochloride buffer was added to the obtained mixture, and they were incubated at room temperature for 10 minutes. 50 xcexcl of a 0.4 mM solution of D-phenylalanyl-L-pipecolyl-L-arginyl-P-nitroanilide dihydrochloride (S-2238, Daiichi Pure Chemicals Co., Ltd.) in pH 8.4 tris hydrochloride buffer was added to the obtained mixture. The absorbance was determined and the initial reaction velocity was calculated. A control was prepared by using 10 xcexcl of tris hydrochloride buffer adjusted to pH 8.4 in place of the solution of the compound to be tested. The absorbance was determined with MICROPLATE READER Model 3550-UV (BIO RAD) at a wavelength of 405 nm at intervals of 15 seconds for 6 minutes. The negative logarithm (pIC50) of the concentration of the sample compound for the 50% inhibition of the activity (initial velocity) of thrombin was determined, and it was employed as the index of the antagonistic activity to thrombin. The antagonistic activities of typical compounds are shown in Table 1 given below.
Thus, the compounds of the present invention have a high antagonistic activity to activated blood coagulation factor VII, and they are useful as pharmaceutical composition for preventing or treating diseases caused by the blood coagulation, thrombus or embolus, intimal thickening or angiostenosis.
Therefore, the compounds of the present invention are useful as pharmaceutical composition for preventing or treating, for example, disseminated intravascular coagulation, deep vein thrombosis, diseases caused by a pulmonary vascular disorder such as pulmonary embolus or pulmonary infarction, diseases caused by an ischemic heart disease such as acute or chronic myocardial infarction or unstable angina, diseases caused by a cerebrovascular disorder such as transient cerebral ischemic attack (TIA), cerebral thrombosis, cerebral infarction, cerebral embolism, cerebral stroke or vasospasm of patients with subarachnoidal bleeding, occlusion of blood vessel and angiostenosis after coronary artery intervention such as Percutaneous transluminal coronary angioplasty (PTCA) or percutaneous transluminal coronary recanalization (PTCR), formation of thrombi after artificial blood vessel-forming operation or artificial valve replacement, peripheral embolism, formation of thrombi in the course of the extracorporeal circulation and antiphospholipid antibody syndrome. When the compounds of the present invention are used for such a purpose, they exhibit a high selectivity toward other enzymes and undesirable side effects such as hemorrhage of patients with serious illness can be advantageously inhibited. Further, since the compounds of the present invention are organic compounds having low molecular weights, they are highly soluble and usable in various dosage forms. The administration routes of them can be selected in a wide range such as intravenous injection and oral administration. The compounds of the present invention are highly stable in blood and very useful.
For the purposes described above, the compounds of the present invention are administered as they are or in the forms of various pharmaceutical compositions. The administration routes of them are, for example, oral administration, parenteral administration such as intravenous, subcutaneous or intramuscular administration, percutaneous administration and rectal administration. The dosage forms of the medicinal compositions are, for example, tablets, suppositories, pills, capsules, powders, suspensions, aerosols, liquids, injections, syrups and emulsions. These medicinal compositions can be prepared by an ordinary preparation method. They comprise a compound of the present invention as the active ingredient and a well-known excipient or carrier and, they contain, if necessary, an adjuvant, additives, etc. For tablets, for example, the adjuvants are inert diluents such as lactose, calcium carbonate and calcium phosphate; the adjuvants are binders such as acacia, corn starch and gelatin, and lubricants such as magnesium stearate, talc and carboxymethyl cellulose; and the additives are sweetening agents such as sucrose, lactose and saccharin, and corrigents such as peppermint and cherry.
The dose of the compounds of the present invention as the anticoagulants varies depending on the administration method, age, body weight and condition of the patient to be treated. The dose is usually 0.01 to 1,000 mg, preferably 0.1 to 50 mg, a day for adults in the oral administration, and 1 xcexcg to 100 mg, preferably 0.01 to 10 mg, a day for adults in the parenteral administration.